The conformation of the nitroxyl spin-label ethyl 3-(2,2,5,5-tetramethylpyrrolinyl-1-oxyl)-propen-2-oate has been determined by electron nuclear double resonance (ENDOR) spectroscopy and computer-based molecular modeling. From ENDOR spectra of the compound in frozen solution, we have assigned resonance absorption features for each class of protons, and we have identified their principal hyperfine coupling (hfc) components from analysis of the dependence of ENDOR spectra on the static laboratory magnetic field. The dipolar hfc components yielded estimates of the electron-proton separations for each class of protons of the ethyl propenoyl moiety. Torsion angle search calculations were carried out to determine the conformational space compatible with hard-sphere nonbonded constraints and with the ENDOR-determined distance constraints. Molecular graphics analysis revealed that the propenoyl side chain of the spin-label exhibits an extended trans conformation and that the ethyl moiety of the ester group deviates significantly from coplanarity with the carboxylate--COO--atoms. The conformation of this molecule is compared with that of an analogous compound O-[3-(2,2,5,5-tetramethylpyrrolinyl-1-oxyl)-propen-2-oyl]-L- beta- phenyllactate, which has been employed as a spectroscopic substrate probe of carboxypeptidase A (L. C. Kuo, J. M. Fukuyama, and M. W. Makinen (1983) Journal of Molecular Biology 163, 63-105). The rotamer conformation of the free spin-label ester in solution, as determined in this study, and that of the enzyme-bound spin-labeled phenyllactate are compared. Differences in rotamer structure are discussed in terms of stereoelectronic principles that govern the pathway of substrate hydrolysis catalyzed by carboxypeptidase A.